Traumatic injury of the spinal cord results in permanent functional impairment. Most of the deficits associated with spinal cord injury result from the loss of axons that are damaged in the central nervous system (CNS). Similarly, other diseases of the CNS are associated with axonal loss and retraction, such as stroke, human immunodeficiency virus (HIV) dementia, prion diseases, Parkinson's disease, Alzheimer's disease, multiple sclerosis and glaucoma. Common to all of these diseases is the loss of axonal connections with their targets, and cell death. The ability to stimulate growth of axons from the affected or diseased neuronal population would improve recovery of lost neurological functions, and protection from cell death can limit the extent of damage. For example, following a white matter stroke, axons are damaged and lost, even though the neuronal cell bodies are alive, and stroke in grey matter kills many neurons and non-neuronal (glial) cells. Treatments that are effective in eliciting sprouting from injured axons are equally effective in treating some types of stroke (Boston life sciences, Sep. 6, 2000 Press release). Neuroprotective agents often tested as potential compounds that can limit damage after stroke. Compounds which show both growth-promotion and neuroprotection are especially good candidates for treatment of stroke and neurodegenerative diseases. Similarly, although the following discussion will generally relate to delivery of Rho antagonists, etc. to a traumatically damaged nervous system, this invention may also be applied to damage from unknown causes, such as during stroke, multiple sclerosis, HIV dementia, Parkinson's disease, Alzheimer's disease, prion diseases or other diseases of the CNS were axons are damaged in the CNS environment. Also, Rho is an important target for treatment of cancer and metastasis (Clark et al (2000) Nature 406:532-535), and hypertension (Uehata et al. (1997) Nature 389:990) and RhoA is reported to have a cardioprotective role (Lee et al. FASEB J. 15:1886-1884). Therefore, the new C3-like proteins are expected to be useful for a variety of diseases were inhibition of Rho activity is required.
It has been proposed to use various Rho antagonists as agents to stimulate regeneration of (cut) axons, i.e. nerve lesions; please see, for example, Canadian Patent application Nos. 2,304,981 (McKerracher et al) and 2,300,878 (Strittmatter). These patent application documents propose the use of known Rho antagonists such as for example C3, chimeric C3 proteins, etc. (see blow) as well as substances selected from among known trans-4-amino (alkyl)-1-pyridylcarbamoylcyclohexane compounds (also see below) or Rho kinase inhibitors for use in the regeneration of axons. C3 inactivates Rho by ADP-ribosylation and is fairly non-toxic to cells (Dillon and Feig (1995) Methods in Enzymology: Small GTPases and their regulators Part. B.256:174-184).
While the following discussion will generally relate or be directed at repair in the CNS, the techniques described herein may be extended to use in many other diseases including, but not restricted to, cancer, metastasis, hypertentension, cardiac disease, stroke, diabetic neuropathy, and neurodegenerative disorders such as stroke, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS). Treatment with Rho antagonists would be used to enhance the rate of axon growth of peripheral nerves and thereby be effective for repair of peripheral nerves after surgery, for example after reattaching severed limbs. Also, treatment with our fusion compounds (proteins) is expected to be effective for the treatment of various peripheral neuropathies because of their axon growth promoting effects.
As mentioned above, traumatic injury of the spinal cord results in permanent functional impairment. Axon regeneration does not occur in the adult mammalian CNS because substrate-bound growth inhibitory proteins block axon growth. Many compounds, such as trophic factors, enhance neuronal differentiation and stimulate axon growth in tissue culture. However, most factors that enhance growth and differentiation are not able to promote axon regenerative growth on inhibitory substrates. To demonstrate that a compound known to stimulate axon growth in tissue culture most accurately reflects the potential for therapeutic use in axon regeneration in the CNS, it is important for the cell culture studies to include the demonstration that a compound can permit axon growth on growth inhibitory substrates. An example of trophic and differentiation factors that stimulate growth on permissive substrates in tissue culture, are neurotrophins such as nerve growth factor (NGF) and brain-derived growth factor. NGF, however, does not promote growth on inhibitory substrates (Lehmann, et al. (1999) 19: 7537-7547) and it has not been effective in promoting axon regeneration in vivo. Brain derived neurotrophic factor (BDNF) is not effective to promote regeneration in vivo either (Mansour-Robaey, et al. J. Neurosci. (1994) 91: 1632-1636). BDNF does not promote neurite growth on growth inhibitory substrates (Lehmann et al supra).
Targeting intracellular signaling mechanisms involving Rho and the Rho kinase for promoting axon regeneration has been proposed (see, for example, the above-mentioned Canadian Patent application nos. 2,304,981 (McKerracher et al)). For demonstration that inactivation of Rho promotes axon regeneration on growth inhibitory substrates, recombinant C3, a protein that inactivates Rho by ADP ribosylation of the effector domain was used. While such a C3 protein can effectively promote regeneration, it has been noted that such a C3 protein does not easily penetrate into cells, and high doses must therefore be applied for it to be effective.
The high dose of recombinant C3 needed to promote functional recovery presents a practical constraint or limitation on the use of C3 in vivo to promote regeneration (Lehmann, et al. (1999) J. Neurosci. 19: 7537-7547; Morii, N and Narumiya, S. (1995) Methods in Enzymology, Vol 256 part B, pg. 196-206. In tissue culture studies, it has, for example, been determined that the minimum amount of C3 that can be used to induce growth on inhibitory substrates is 25 ug/ml (Lehmann, et al. (1999) J. Neurosci. 19: 7537-7547; Morii, N and Narumiya, S. (1995) Methods in Enzymology, Vol 256 part B, pg. 196-206. If the cells are not triturated, even this dose is ineffective. It has been estimated, for example, that at least 40 μg of C3 per 20 g mouse needs to be applied to injured mouse spinal cord or rat optic nerve (McKerracher, Canadian patent application No.: 2,325,842). Calculating doses that would be required to treat an adult human on an equivalent dose per weight scale up used for rat and mice experiments, it would be necessary to apply 120 mg/kg of C3 (i.e. alone) to the injured human spinal cord. The large amount of recombinant C3 protein needed creates significant problems for manufacturing, due to the large-scale protein purification and cost. It also limits the dose ranging that can be tested because of the large amount of protein needed for minimal effective doses.
Another related limitation with respect to the use of C3 to promote repair in the injured CNS is that it does not easily penetrate the plasma membrane of living cells. In tissue culture studies when C3 is applied to test biological effects it has been microinjected directly into the cell (Ridley and Hall (1992) Cell 70: 389-399), or applied by trituration of the cells to break the plasma membrane (Lehmann, et al. (1999) J. Neurosci.19: 7537-7547, Jin and Strittmatter (1997) J. Neurosci. 17: 6256-6263). In the case of axon injury in vivo, the C3 protein is likely able to enter the cell because injured axons readily take up substances from their environment. However, C3-like proteins of the present invention are likely to act also on surrounding undamaged neurons and help them make new connections as well, thus facilitating recovery. After incomplete SCI, there is plasticity of motor systems attributed to cortical and subcortical levels, including spinal cord circuitry (Raineteau, O., and Schwab, M. E. (2001) Nat Rev Neurosci 2: 263-73). This plasticity may be attributed to axonal or dendritic sprouting of collaterals and synaptic strengthening or weakening. Additionally, it has been shown that sparing of a few ventrolateral fibers may translate into significant differences in locomotor performance since these fibers are important in the initiation and control of locomotor pattern through spinal central pattern generators (Brustein, E., and Rossignol, S. (1998) J Neurophysiol 80: 1245-67). It is well documented that reorganization of spared collateral cortical spinal fibers occurs after spinal cord injury and this contributes to functional recovery (Weidner et al, 2001 Proc. Natl. Acad. Sci. 98: 3513-3518). The process of reorganization and sprouting of spared fibers would be enhanced by treatment with C3-like proteins able to enter non-injured neurons. This would enhances spontaneous plasticity of axons and dendritic remodeling known to help functional recovery.
Other methods of delivery of C3 in vitro have been to make a recombinant protein that can be taken up by a receptor-mediated mechanism (Boquet, P. et al. (1995) Meth. Enzymol. 256: 297-306). The disadvantage of this method is that the cells needing treatment must express the necessary receptor. Lastly, addition of a C2II binding protein to the tissue culture medium, along with a C21N-C3 fusion toxin allows uptake of C3 by receptor-mediated endocytosis (Barthe et al. (1998) Infection and Immunity 66:1364). The disadvantage of this system is that much of the C3 in the cell will be restrained within a membrane compartment. More importantly, two different proteins must be added separately for transport to occur (Wahl et al. 2000. J. Cell Biol. 149:263), which make this system difficult to apply to for treatment of disease in vivo.